1260 infinity 2 hplc system

One gram of LR powder was accurately weighed and placed in a 50 mL conical flask. Then 20 mL of methanol solution was added for 30 min ultrasonic extraction, and the liquid was cooled and weighed. Swertiamarin (2 mg), sweroside (1 mg), hesperetin (1 mg), coumarin (4.9 mg), 1.7-dihydroxy-3,8-dimethoxyl xanthone (1 mg), and 1-hydroxy-2,3,5 trimethoxanthone (1 mg) were carefully weighed and put into a 10 mL flask, dissolved in methanol and diluted to scale, shaken well, and filtered through a 0.45 µm microporous filter membrane (each 1 mL contained 0.2 mg swertiamarin, 0.1 mg sweroside, 0.1 mg hesperidin, 0.49 mg coumarin, 0.1 mg 1,7-dihydroxy-3,8-dimethoxone, and 0.1 mg 1-hydroxy-2,3,5-trimethoxone). High performance liquid chromatography (HPLC) analysis was performed on an Agilent 1260 InfinityII HPLC system. ZORBAX SB-C18 5-Micron column (4.6 mm × 250 mm) with mobile phase water (A) - 0.1% phosphate aqueous solution (B) and gradient elution (0-15 min, 30%-35% B; 15 to 25 min, 35%-50% B; 25 to 35 min, 50%-65% B; 35 to 45 min, 65%-70% B; 45 to 50 min, 70%-80% B; 50 to 55 min, 80%-95%; 55-60 min, 95%-100%). The flow rate was set to 1.0 mL/min, the column temperature was set to 30 °C, and the detection wavelength was set at 234 nm. Standards of six compounds were purchased (Sigma-Aldrich, St. Louis, MO, United States), and calibration curves were performed.